The ultimate goal of this proposal is to generate, maintain, and distribute a new collection of duplication stocks that covers nearly the entire euchromatic autosomal portion of the Drosophila melanogaster genome. Since this collection will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists, t is likely that there will be a high demand for this resource. Many of the most important advances in our understanding of human development have come from studies using the fruit fly Drosophila melanogaster as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology. We propose to generate a new duplication collection that, together with existing collections for the X and 4th chromosomes, will cover 99.7% of the entire Drosophila genome. This new duplication set supports a broad range of applications across different fields. First, it will allow rapid mapping and identification of new genes required for normal development and function. Compared to the current mapping approach that uses the molecularly defined Drosophila autosomal deletion kit (MDADK), this duplication collection will offer an average of 10-fold higher resolution. As this duplication kit is based on generating transgenic flies carrying genomic constructs, further manipulation of the same constructs can be performed for downstream functional analysis, including tagging of proteins and structure-function studies. Moreover, these stocks can be used for genetic rescue experiments that are essential for assigning gene function as well as testing and validating the huge collection of mutants held in stocks centers around the world. We estimate that a total of 3,315 transgenic stocks will be needed to complete this collection and we split this project into two phases: Phase I has been funded and is nearly complete: more than 350 of the proposed 400 BACs have been injected and 263 transgenics isolated. Here in Phase II, we will complete the collection. There are three Specific Aims: Aim 1. Select and validate a tiled array of BACs covering chromosomes 2 and 3. Aim 2. Generate and validate a complete autosomal duplication kit. Aim 3. Create and maintain an online searchable database for BACs and transgenics. Success in Phase I has filled 74% of the bases in gaps in the current MDADK, and has also set the stage for expanding our effort to generate the complete collection. Harnessing the benefits of new technologies, this collection will be another key addition to the Drosophila research tool kit. As a highly desired resource by most Drosophila geneticists across the world, this collection will offer great utility or the entire Drosophila research community, with applications ranging from genetic fine mapping, genomic rescue, somatic clonal analysis, and genomic tagging.